A Novel LC-MS/MS Method for Simultaneous Determination of Ivabradine and its Active Metabolite N-Desmethyl Ivabradine in Human Plasma: Its Pharmacokinetic Application

The method was new simple, rapid, sensitive and simultaneous liquid chromatography/tandem mass spectrometry assay method for the determination of Ivabradine and N-Desmethyl Ivabradine in human plasma using Ivabradine-d 6 and N-Desmethyl Ivabradine-d 6 as internal standards (IS).Analyte and the internal standards were extracted from the human plasma via Solid phase extraction (SPE) using Strata TM X 33µM polymeric sorbent cartridges (1cc/30mg).The chromatographic separation was achieved on a Kromasil 100-5 C 18 , 100 x 4.6 mm, 5 µm columns by using a gradient programme at a flow rate of 0.60 mL/min with a total runtime of 3.0 min and the elution was monitored by multiple reaction monitoring modes using electropspray ionization.The calibration curve obtained was linear (r 2  0.99) over the concentration range of 0.20-201 ng/mL for Ivabradine and 0.10-15.14for N-Desmethyl Ivabradine.Method validation was performed as per FDA guidelines and the results met the within the acceptable limits.The proposed method was found to be applicable to pharmacokinetic studies.